Our investigations of the structure and function of the tryptophan synthase multienzyme complex are providing insights into mechanisms of catalysis, metabolite channeling, and allosteric interactions. We have used cryo-crystallography to determine the structure of a mutant (alphaD60N) tryptophan synthase alpha2/beta2 complex with the true substrate, indole glycerol phosphate, bound to the active site of the alpha subunit. The structure reveals the correct orientation of the active site Glu49 and supports the proposed role of Glu49 in catalysis. Cryo-crystallography and microspectrophotometry of this mutant enzyme with L-serine bound to the beta subunit and indole propanol phosphate bound to the alpha subunit reveals allosteric roles of alphaAsp60. We have used information from the crystal structure to design site-directed mutagenesis experiments to probe the relationships between structure and function. Mutation of a residue in the cofactor binding site of tryptophan synthase (beta-Serine377) alters the cofactor chemistry and leads to mechanism-based inactivation. Mutation of residues in the interaction site between the alpha and beta subunits alters the transmission of allosteric signals between the two active sites of the two subunits.